Laser Phototherapy at High Energy Densities Do Not Stimulate Pre-Osteoblast Growth and Differentiation

Abstract

Objective: The aim of this study is to evaluate the effects of red and infrared lasers at high energy densities on pre-osteoblast MC3T3 proliferation and differentiation. Background data: The acceleration of bone regeneration by low intensity laser irradiation may hold potential benefits in clinical therapy in orthopedics and dentistry. Materials and methods: Cells were irradiated with red (660 nm) and infrared (780 nm) lasers (90 and 150 J/cm², 40 mW). The control group did not receive irradiation. Cell growth was assessed by a colorimetric test (MTT) (24, 48, 72, 96 h) and cell differentiation was evaluated by alkaline phosphatase (ALP) quantification after growth in osteogenic medium (72, 96 h; 7, 14 days). Results: None of the irradiation groups had an enhancement in cell growth (p<0.05). The production of ALP was not influenced by irradiation at any period of time (p>0.05). Conclusions: The low intensity laser stimulated neither cell growth nor the production of alkaline phosphatase.

Introduction

P hototherapy with low intensity lasers has been used to improve wound healing. The precise biochemical mechanism underlying the therapeutic effects of phototherapy is not yet well established.

From observation, it appears that it has a wide range of effects at the molecular, cellular, and tissue levels.⁹ Some studies showed stimulus on cell proliferation,² growth factor release,³ protein synthesis,⁴ and vasodilation.⁵ However, the literature about laser effects is controversial. Research in humans failed to show that phototherapy stimulated wound healing.^6
–8

The acceleration of bone cell proliferation and differentiation by lasers may be of great value in medicine and dentistry. Some studies found higher proliferation rates and bone nodule formation after low intensity laser irradiation.^9,10 Also, increased alkaline phosphatase (ALP) activity and osteocalcin expression were observed, suggesting direct stimulatory effects on bone formation by laser irradiation.¹⁰

The parameters of laser application are so varied in the literature that it maks any comparison between research studies difficult. It was suggested that high energy densities caused a delay in wound healing.¹¹ Thus, if the energy density and irradiation time are too high, the response may be inhibited.¹

There are a few studies concerning the effects of high energy densities of laser on wound healing, mainly on osteoblasts. Therefore, the purpose of this study is to evaluate the effects of red and infrared laser at high energy densities on pre-osteoblast MC3T3 proliferation and differentiation.

Material and Methods

Pre-osteoblasts (MC3T3–ATCC–E1 subclone 4) were cultured in modified Eagle's medium (MEM) complemented by eight nucleosides (adenosine, cytidine, deoxyadenosine, deoxycytidine, deoxyguanosine, guanosine, thymidine, and viridine),¹² supplemented by 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution in a humidified air–5% carbon dioxide (CO₂) atmosphere. Cells of the 17th passage were used for the experiments.

Experimental groups

The experimental groups were divided according to treatments as:

C+= positive control (medium plus 10% FBS)

LASIR90=infrared laser – 90 J/cm²

LASIR150=infrared laser – 150 J/cm²

LASR90=red laser – 90 J/cm²

LASR150=red laser – 150 J/cm²

Laser irradiation

Laser irradiation was delivered with indium–gallium–aluminum phosphide/gallium–aluminum–arsenium (InGaAlP/GaAlAs) diode lasers (MMOptics Ltd., São Carlos, SP, Brazil). Irradiations were performed in contact with the bottom of the plate, using the punctual irradiation mode, in a 0.042 cm² area. Two wavelengths were used: 660 nm (visible red) and 780 nm (infrared), with powers of 40±6.24 mW and power density of 1 W/cm² for both wavelengths. Two doses (energy density) settings were used: 90 and 150 J/cm² (90 and 150 sec, respectively).

A plate for each treatment (control, red laser, and infrared laser) was used. During irradiation, the wells of the other experimental groups were protected by a black mask to avoid additional irradiation. Control non-irradiated cells were maintained outside the incubator under the same condition as the laser-irradiated cells.

The optical output power of the light sources was checked before the experiments with the use of a power meter (FieldMax II, Coherent, Santa Clara, CA).

Cell growth

Twenty-four 96-well microtitration plates were used. On the 1st day of the experiment, 10³ cells were plated into each well. On the 2nd day, the medium was substituted with a quiescent environment of MEM plus nucleosides supplemented with 1% FBS, for another 24 h. This culturing period was necessary in order to synchronize the cell cycle. On the 3rd day, the irradiated and positive control groups received a fresh 10% osteogenic medium (MEM+nucleosides) supplemented by 2.5 mg of ascorbic acid and 50 μg/mL of glicerophosphate. Immediately afterwards, the experimental groups received irradiation with laser. For the next 4 days, every 24 h, one plate was submitted to the MTT assay to obtain data for the cell growth pattern. The MTT assay measures cell mitochondrial activity. This assay involves the conversion of the water-soluble MTT to an insoluble formazan. The formazan is then solubilized, and the concentration determined by optical density at≅570 nm.

ALP activity

Twenty-four 24-well plates were used and 4×10⁴ cells were plated into each well. The first 3 days of the experiment were the same as described previously, with the difference being the use of the scanning mode of application of laser. The cell lysate was collected after 72 h, 96 h, 7 days, and 14 days. The culture medium was removed, the cells were rinsed in phosphate-buffered saline solution, lysed in 250 μL 25 mM Tris-phosphate (pH 7.8) buffer containing 1% Triton-X, and kept frozen until required for the assessment of ALP activity. ALP was assayed at pH 10.0 using p-nitrophenyl phosphate as substrate (pNP). The reaction mixture (0.5 mL) contained pNP (10 mM), MgCl2 (2 mM), 2-amino-2-methylpropanol/HCl buffer (0.5 M), and an appropriate amount of extract. Incubation was at 37°C for 15 or 30 min, after which time 0.5 mL of 0.75N NaOH was added to each well. Absorbancies were measured at 405 nm. One unit of ALP is that quantity of enzyme that releases 1 μmol of p-nitrophenol per hour. Specific activities are given as units per milligram of protein (U/mg). Protein was determined by the method of Bradford.¹³

Statistical analysis

The statistical analysis was performed using the repeated measures analysis of variance test complemented by the Tukey test, with a confidence interval of 95% (p<0.05).

Results

Cell growth

At 24 h, the mean optic density varied from 0.079 to 0.135 (p<0.05) (Table 1). At 48 h, mean optic density varied from 0.085 to 0.173. At 72 h, mean optic density varied from 0.103 to 0.180. At 96 h, mean optic density varied from 0.161 to 0.246. There were no statistically significant differences among groups for 24, 48, 72, and 96 h (p>0.05) (Table 1).

Table 1.

Cell Growth per Period of Time (Optic Density)

	24 h		48 h		72 h		96 h
Period	Mean	SD	Mean	SD	Mean	SD	Mean	SD
C+	0.103	0.052	0.104	0.044	0.103	0.049	0.161	0.016
LASIR90	0.135	0.037	0.173	0.087	0.103	0.029	0.230	0.021
LASIR150	0.098	0.014	0.094	0.053	0.178	0.065	0.228	0.033
LASR90	0.092	0.018	0.111	0.047	0.180	0.041	0.246	0.040
LASR150	0.079	0.017	0.085	0.017	0.147	0.09	0.236	0.020

Data not statistically significant (p>0.05).

C+, positive control (medium plus 10% fetal bovine serum); LASIR90, infrared laser – 90 J/cm²; LASIR150, infrared laser – 150 J/cm²; LASR90, red laser – 90 J/cm²; LASR150, red laser – 150 J/cm².

ALP activity

At 72 h, the ALP activity varied from 0.108 to 0.191 μmol pNP/min×mg (Table 2). At 96 h, it varied from 0.078 to 0.139 μmol pNP/min×mg (Table 2). At 7 days, it varied from 0.063 to 0.106 μmol pNP/min x mg (Table 2). At 14 days, it varied from 0.031 to 0.039 μmol pNP/min × mg (Table 2). There were no statistically significant differences between groups (p>0.05).

Table 2.

Alkaline Phosphatase (μMol pNP/Min×mg) Release by Cells per Period of Time

	72 h		96 h		7 days		14 days
Period	Mean	SD	Mean	SD	Mean	SD	Mean	SD
C+	0.163	0.034	0.139	0.052	0.106	0.027	0.039	0.006
LASIR90	0.182	0.061	0.111	0.009	0.096	0.005	0.031	0.005
LASIR150	0.191	0.085	0.085	0.042	0.068	0.020	0.034	0.009
LASR90	0.108	0.016	0.078	0.007	0.095	0.021	0.033	0.004
LASR150	0.108	0.016	0.079	0.003	0.063	0.016	0.031	0.004

Data not statistically significant (p>0.05).

Discussion

In the present research, we evaluated the cell growth and ALP activity by MC3T3 cells after irradiation with red and infrared laser with high doses of 90 and 150 J/cm². The energy densities were chosen based on an article that showed a delayed wound healing after irradiation with a red laser (632.8 nm) with 20 J/cm².¹¹ We could not observe an impaired cell growth or ALP production, only a lack of effect of laser irradiation. Another study tested the effects of red laser 660nm (150 and 1050 J/cm²) on fibroblasts.¹⁴ The group that received 1050 J/cm² at 48 h presented a delay in cell growth and higher rates of cell death. The authors suggested a dose dependency that can be determined from the dosage used and the time of irradiation. In cell cultures, any changes in the irradiation protocol can induce alterations in the outcome.¹⁴ In clinical studies of chronic tendinopathy, high doses of laser (21 J) have yielded poor results.¹⁵ On the other hand, a study of liver regeneration analysis in rats described a stimulation effect with a red laser with 22.5 J/cm² of energy density.¹⁶ The same positive results were obtained using an infrared laser with 1589 J/cm² for axonal regeneration and functional recovery of spinal cord injury.¹⁷ Chung et al. published a revision affirming that if the light applied is not of sufficient irradiance or the irradiation time is too short, then there is no response.¹ If the irradiance is too high or irradiation time is too long, then the response may be inhibited.¹

Studies that used low energy densities presented beneficial effects of laser. Fujihara et al. showed a proliferative stimulus on osteoblast-like cells with red laser (3 J/cm²).² Abramovitch-Gottlib et al. found higher ossification levels in irradiated samples with a red HeNe (632.8 nm) laser (0.5 mW/cm²).¹⁸ Laser stimulated the differentiation of mesenchymal stem cells into osteoblasts on a three-dimensional coralline biomatrix.¹⁸ There was an enhancement on tissue formation, appearance of phosphorus peaks and calcium and phosphate incorporation to newly formed tissue and production of ALP.¹⁸

The choice of light source and dosage is particularly important, as there is an optimal dose of light for any particular application, and doses higher or lower than this optimal value may have no therapeutic effect. Phototherapy is characterized by a biphasic dose response: lower doses of light are often more beneficial than high doses¹.

In our study, ALP activity was analyzed at 72 h, 96 h, 7 days, and 14 days, based on previous studies that showed that ALP activity peaks at the end of the proliferative stage and before matrix maturation.¹⁹ Osteogenically induced cells show a peak of ALP activity between ∼7 days and 14 days after they have been seeded in the osteogenic differentiation medium.²⁰ Our study, on the other hand, showed a peak at 96 h (0.456 μmol pNP/min×mg) that decreased until 14 days (0.059 μmol pNP/min×mg). One possible explanation for these results is the reaching of confluence at an earlier point of time. The cells of a confluent monolayer have the tendency to inhibit growth and finally die when they are not subcultured in time.²¹

Many studies showed an increase in ALP activity after laser irradiation.^22
–24 Haxsen et al. performed a study in which osteoblasts were irradiated with a red laser 690 nm, 51, 102, or 204 mW/cm², every 4 h for 24 h in total.²² The 102 mW/cm² irradiance promoted a 43% increase in ALP production, and the irradiance of 204 mW/cm² increased the production of ALP eightfold.²² These results differ from ours, in which there was no increase in ALP activity with laser.²² The study by Hirata et al. is in accordance with our research.²³ They examined the effects of irradiation by a GaAlAs (805 nm) infrared laser on bone morphogenetic protein (BMP)2-induced ALP activity in C2C12 cells, which differentiate into osteoblasts upon treatment with BMP2.²³ When the cells were treated with laser irradiation at 0.5 W for 20 min together with BMP2, ALP activity was significantly increased.²³ However, laser treatment alone did not affect ALP activity.²³ A possible explanation for the lack of laser effects is that the irradiations were performed once. Therefore, the effects of irradiation are limited to a small period of time. Horvát-Karajz et. al. stated that biostimulation after irradiation continues for 48 h²⁵ and after this time, the effects may no longer occur. Some authors showed an enhancement of cell proliferation and alkaline phosphatase production after multiple irradiations.^22,26

The lack of results of this study could be attributed to the methodology of irradiation employed. In the first experiment, we used a 96-well plate and each plate had a diameter of 6.35 mm. Because the tip of the laser had a diameter of 4.2 mm, we assumed that only 66% of the cells were irradiated. However, in our recent article,²⁷ we used the same methodology with lower energy densities, and the cell growth was stimulated by laser, irradiating only 66% of the cells. In the second experiment, we used a 24-well plate that had a 15.62 mm diameter. In order to irradiate all cells, we used a scanning mode of laser application. Therefore, we suggest that the higher energy densities were responsible for the lack of results.

Conclusions

In conclusion, low intensity red and infrared laser at 90 and 150 J/cm² did not stimulate pre-osteoblast cell growth and the alkaline phosphatase production.

Footnotes

Acknowledgments

The authors thank Heitor Marques Honório for statistical analysis. This research was supported by a grant from Programa Institucional de Bolsas de Iniciação Científica (PIBIC) - Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) # 2011-316.

Author Disclosure Statement

No competing financial interests exist.

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